CULTURES OF CHYTRIDS - PARASITES OF ALGAE IN BIOLOGICAL INSTITUTE OF PETERSBURG UNIVERSITY

Although diatom extracellular matricies are usually thought of exclusively in terms of the beautiful, architecturally complex silicious frustule, polymers exuded through the frustule are critical mediators of interactions with the external environment. In several species, complex proteoglycans appear to be the primary components involved in adhesion and motility. When viewed with high-resolution cryo-scanning electron microscopy methods, the ubiquity and pervasiveness of these polymers was revealed in both freshwater and marine taxa. Monoclonal antibody mapping of carbohydrate epitopes characterized by NMR, methylation and monosaccharide analysis and correlated with structural observations by EM revealed an organizational pattern far more complex than previously proposed. Modeling assembly of extracellular "stalks" in the marine biofouling diatom Achnanthes longipes involves intracellular sequestering of multiple components, deposition at the protoplasmic membrane/diatotepum interface, transport through the multilayered diatotepum and holes in the silica, extrusion from the frustule, and assembly into a very complex multi-laminate biocomposite structure. The mechanism of extracellular polymer participation in motility is complex in a different way, as some current models of raphe associated motility involve cytoskeletal interactions and molecular motors.     

Extracellular matrix assembly in diatoms (Bacillariophyceae). iv. ultrastructure of Achnanthes longipes and Cymbella cistula as revealed by high-pressure freezing/freeze substituton and cryo-field emission scanning electron microscopy

Extracellular matrix (ECM) polymers secreted by the diatoms Achnanthes longipes Ag. and Cymbella cistula (Ehr.) Kirchn. completely encase the cell and are responsible for adhesion and other interactions with the external environment. To preserve details of the highly hydrophilic ECM in the native state and to preserve, with a high degree of fidelity, the intracellular structures involved in synthesis of extracellular polymers, we applied a suite of cryotechniques. The methods included high‐resolution visualization of surfaces using cryo‐field emission SEM (cryo‐FESEM) and preservation for TEM observation of thin sections by high‐pressure freezing (HPF) and freeze substitution (FS). The extracellular structures of diatoms plunge‐frozen in liquid ethane, etched at low temperature, and observed on a cryostage in the FESEM showed overall dimensions and shapes closely comparable to those observed with light microscopy. Cryo‐FESEM demonstrated the pervasive nature of the extracellular polymers and their importance in cell–substratum and cell–cell associations and revealed details of cell attachment processes not visible using other SEM techniques or light microscopy. The layer of ECM coating the frustule and entirely encapsulating cells of A. longipes and C. cistula was shown to have a significant role in initial cell adhesion and subsequent interaction with the environment. Trails of raphe‐associated ECM, generated during cell motility, were shown at high resolution and consist of anastomoses of coiled and linear strands. Cryo‐FESEM revealed a sheet‐like mucilage covering stalks. HPF/FS of A. longipes resulted in excellent preservation of intra‐ and extracellular structures comparable to previous reports for animals and higher plants and revealed several organelles not described previously. Three distinct vesicle types were identified, including a class closely associated with Golgi bodies and postulated to participate in formation of the extracellular adhesive structures. HPF/FS showed a number of continuous diatotepic layers positioned between the plasma membrane and the silicon frustule and revealed that extracellular adhesive extrusion through frustule pores during stalk production was closely related to the diatotepum. The stalks of A. longipes consist of highly organized, multilayered, fine fibrillar materials with an electron‐opaque layer organized as a sheath at the stalk periphery.     

Micro‐photoluminescence of single living diatom cells

Diatoms are single‐celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro‐photoluminescence (μ‐PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500–510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto‐fluorescence of photosynthetic pigments. The portion of the μ‐PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ‐PL spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.     

NANOSTRUCTURE OF THE DIATOM FRUSTULE AS REVEALED BY ATOMIC FORCE AND SCANNING ELECTRON MICROSCOPY

The cell wall (frustule) of the freshwater diatom Pinnularia viridis (Nitzsch) Ehrenberg is composed of an assembly of highly silicified components and associated organic layers. We used atomic force microscopy (AFM) to investigate the nanostructure and relationship between the outermost surface organics and the siliceous frustule components of live diatoms under natural hydrated conditions. Contact mode AFM imaging revealed that the walls were coated in a thick mucilaginous material that was interrupted only in the vicinity of the raphe fissure. Analysis of this mucilage by force mode AFM demonstrated it to be a nonadhesive, soft, and compressible material. Application of greater force to the sample during repeated scanning enabled the mucilage to be swept from the hard underlying siliceous components and piled into columns on either side of the scan area by the scanning action of the tip. The mucilage columns remained intact for several hours without dissolving or settling back onto the cleaned valve surface, thereby revealing a cohesiveness that suggested a degree of cross-linking. The hard silicified surfaces of the diatom frustule appeared to be relatively smooth when living cells were imaged by AFM or when field-emission SEM was used to image chemically cleaned walls. AFM analysis of P. viridis frustules cleaved in cross-section revealed the nanostructure of the valve silica to be composed of a conglomerate of packed silica spheres that were 44.8 ± 0.7 nm in diameter. The silica spheres that comprised the girdle band biosilica were 40.3 ± 0.8 nm in diameter. Analysis of another heavily silicified diatom, Hantzschia amphioxys (Ehrenberg) Grunow, showed that the valve biosilica was composed of packed silica spheres that were 37.1 ± 1.4 nm and that silica particles from the girdle bands were 38.1 ± 0.5 nm. These results showed little variation in the size range of the silica particles within a particular frustule component (valve or girdle band), but there may be differences in particle size between these components within a diatom frustule and significant differences are found between species.     

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (II. 2,6-Dichlorobenzonitrile Inhibition of Motility and Stalk Production in the Marine Diatom Achnanthes longipes)

The cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB) and the DCB analogs 2-chloro-6-fluorobenzonitrile, 3-amino-2,6-dichlorobenzonitrile, and 5-dimethylamino-naphthalene-1-sulfonyl-(3-cyano-2, 4-dichloro)aniline (DCBF) inhibited extracellular adhesive production in the marine diatom Achnanthes longipes, resulting in a loss of motility and a lack of permanent adhesion. The effect was fully reversible upon removal of the inhibitor, and cell growth was not affected at concentrations of inhibitors adequate to effectively interrupt the adhesion sequence. Video microscopy revealed that the adhesion sequence was mediated by the export and assembly of polymers, and consisted of initial attachment followed by cell motility and eventual production of permanent adhesive structures in the form of stalks that elevated the diatom above the substratum. A. longipes adhesive polymers are primarily composed of noncellulosic polysaccharides (B.A. Wustman, M.R. Gretz, and K.D. Hoagland [1997] Plant Physiol 113: 1059-1069). These results, together with the discovery of DCB inhibition of extracellular matrix assembly in noncellulosic red algal unicells (S.M. Arad, O. Dubinsky, and B. Simon [1994] Phycologia 33: 158-162), indicate that DCB inhibits synthesis of noncellulosic extracellular polysaccharides. A fluorescent probe, DCBF, was synthesized and shown to inhibit adhesive polymer production in the same manner as DCB. DCBF specifically labeled an 18-kD polypeptide isolated from a membrane fraction. Inhibition of adhesion by DCB and its analogs provides evidence of a direct relationship between polysaccharide synthesis and motility and permanent adhesion.     

A Widely Distributed Thraustochytrid Parasite of Diatoms Isolated from the Arctic Represents a gen. and sp. nov.

A unicellular, heterotrophic, eukaryotic parasite was isolated from nearshore Arctic marine sediment in association with the diatom Pleurosigma sp. The parasite possessed ectoplasmic threads that could penetrate diatom frustules. Healthy and reproducing Pleurosigma cultures would begin to collapse within a week following the introduction of this parasite. The parasite (2–10 μm diameter) could reproduce epibiotically with biflagellate zoospores, as well as binary division inside and outside the diatom host. While the parasite grew, diatom intracellular content disappeared. Evaluation of electron micrographs from co‐cultures revealed the presence of hollow tubular processes and amorphic cells that could transcend the diatom frustule, generally at the girdle band, as well as typical thraustochytrid ultrastructure, such as the presence of bothrosomes. After nucleotide extraction, amplification, and cloning, database queries of DNA revealed closest molecular affinity to environmental thraustochytrid clone sequences. Testing of phylogenetic hypotheses consistently grouped this unknown parasite within the Thraustochytriidae on a distinct branch within the environmental sequence clade Lab19. Reclassification of Arctic high‐throughput sequencing data, with appended reference datasets that included this diatom parasite, indicated that the majority of thraustochytrid sequences, previously binned as unclassifiable stramenopiles, are allied to this new isolate. Based on the combined information acquired from electron microscopy, life history, and phylogenetic testing, this unknown isolate is described as a novel species and genus.     

On the role of cell surface associated,mucin-like glycoproteins in the pennate diatom Craspedostauros australis (Bacillariophyceae)

Diatoms are single-celled microalgae with silica-based cell walls (frustules) that are abundantly present in aquatic habitats, and form the basis of the food chain in many ecosystems. Many benthic diatoms have the remarkable ability to glide on all natural or man-made underwater surfaces using a carbohydrate- and protein-based adhesive to generate traction. Previously, three glycoproteins, termed FACs (F rustule A ssociated C omponents), have been identified from the common fouling diatom Craspedostauros australis and were implicated in surface adhesion through inhibition studies with a glycan-specific antibody. The polypeptide sequences of FACs remained unknown, and it was unresolved whether the FAC glycoproteins are indeed involved in adhesion, or whether this is achieved by different components sharing the same glycan epitope with FACs. Here we have determined the polypeptide sequences of FACs using peptide mapping by LC–MS/MS. Unexpectedly, FACs share the same polypeptide backbone (termed CaFAP1), which has a domain structure of alternating Cys-rich and Pro-Thr/Ser-rich regions reminiscent of the gel-forming mucins. By developing a genetic transformation system for C. australis, we were able to directly investigate the function of CaFAP1-based glycoproteins in vivo. GFP-tagging of CaFAP1 revealed that it constitutes a coat around all parts of the frustule and is not an integral component of the adhesive. CaFAP1-GFP producing transformants exhibited the same properties as wild type cells regarding surface adhesion and motility speed. Our results demonstrate that FAC glycoproteins are not involved in adhesion and motility, but might rather act as a lubricant to prevent fouling of the diatom surface.     

EXTRACELLULAR MATRIX ASSEMBLY IN DIATOMS (BACILLARIOPHYCEAE). V. ENVIRONMENTAL EFFECTS ON POLYSACCHARIDE SYNTHESIS IN THE MODEL DIATOM,PHAEODACTYLUM TRICORNUTUM1

The effects of phosphate (P) limitation, varying salinity (5–65 psu), and solid media growth conditions on the polysaccharides produced by the model diatom, Phaeodactylum tricornutum Bohlin were determined. Sequential extraction was used to separate polymers into colloidal (CL), colloidal extracellular polymeric substances (cEPS), hot water soluble (HW), hot bicarbonate soluble (HB), and hot alkali (HA) soluble fractions. Media‐soluble polymers (CL and cEPS) were enriched in 4‐linked mannosyl, glucosyl, and galactosyl residues as well as terminal and 3‐linked xylosyl residues, whereas HW polymers consisted mainly of 3‐linked glucosyl as well as terminal and 2,4‐linked glucuronosyl residues. The HB fraction was enriched in terminal and 2‐linked rhamnosyl residues derived from the mucilage coating solubilized by this treatment. Hot alkali treatment resulted in the complete dissolution of the frustule releasing 2,3‐ and 3‐linked mannosyl residues. The fusiform morphotype predominated in standard and P‐limited cultures and cultures subjected to salinity variations, but growth on solid media resulted in an enrichment of the oval morphotype. The proportion and linkages of 15 residues, including neutral, uronic acid, and O‐methylated sugars, varied with environmental conditions. P limitation and salinity changes resulted in 1.5‐ to 2.5–fold increase in carbohydrate production, with enrichment of highly branched/substituted and terminal rhamnose, xylose, and fucose as well as O‐methylated sugars, uronic acids, and sulfate. The increased deoxy‐ and O‐methylated sugar content under unfavorable environments enhances the hydrophobicity of the polymers, whereas the anionic components may play important roles in ionic cross‐linking, suggesting that these changes could ameliorate the effects of salinity or P‐stress and that these altered polysaccharide characteristics may be useful as bioindicators for environmental stress.     

Use of fluorophore-conjugated lectins to study cell-cell interactions in model marine biofilms

Biofilms dominated by pennate diatoms are important in fields as diverse as ship biofouling and marine littoral sediment stabilization. The architecture of a biofilm depends on the fact that much of its mass consists of extracellular polymers. Although most illuminated biofilms in nature are dominated by phototrophs, they also contain heterotrophic bacteria. Given the close spatial association of the two types of organisms, cell-cell interaction is likely. Fluorophore-conjugated lectins were used to demonstrate the sites of the various extracellular polymers in three species of diatoms. Based on their lectin staining properties, the polymers in different species appeared to be similar, but their involvement in the process of attachment to a surface differed. In a coculture Pseudoalteromonas sp. strain 4 or its sterilized spent medium reduced the ability of Amphora coffeaeformis and Navicula sp. strains 1 and D to adhere, inhibited motility, and caused agglutination and eventually diatom cell lysis. Diatoms could be protected from the negative effects of the bacterial spent medium if D-galactose or mannan was included in the incubation medium. The active principle of the spent medium is probably a lectin/agglutinin that is able to bind to the extracellular polymers of the diatoms that are involved in adhesion and motility. Awareness of interactions of this type is important in the study of natural biofilms.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.     

OBSERVATIONS ON THE ULTRASTRUCTURE OF THE MALI GAMETES OF BIDDULPHIA LEVIS EHR.1

The marine centric diatom Biddulphia levis produced uniflagellate fusiform male gametes completely within the parent cell frustule. These gametes lacked both a central pair of microtubules in the flagellar axoneme and chloroplasts but did contain a cone of microtubules which passed posteriorly from the base of the kinetosome along the nuclear envelope. The gametes were released through a specialized pore in the girdle band leaving behind a cytoplasmic mass which contained chloroplasts and other cytoplasmic components. Tubules which resembled the flimmer hairs on the gamete flagellum occurred in cisternae of the cytoplasmic reticulum in the residual cytoplasm and in the nuclear envelope of the gametes. Gametogenesis in B. levis is compared with similar processes in other centric diatoms.     

Shaping the phycosphere: Analysis of the EPS in diatom-bacterial co-cultures

The phycosphere is a unique niche that fosters complex interactions between microalgae and associated bacteria. The formation of this extracellular environment, and the associated bacterial biodiversity, is heavily influenced by the secretion of extracellular polymers, primarily driven by phototrophic organisms. The exopolysaccharides (EPS) represent the largest fraction of the microalgae-derived exudates, which can be specifically used by heterotrophic bacteria as substrates for metabolic processes. Furthermore, it has been proposed that bacteria and their extracellular factors play a role in both the release and composition of the EPS. In this study, two model microorganisms, the diatom Phaeodactylum tricornutum CCAP 1055/15 and the bacterium Pseudoalteromonas haloplanktis TAC125, were co-cultured in a dual system to assess how their interactions modify the phycosphere chemical composition by analyzing the EPS monosaccharide profile released in the culture media by the two partners. We demonstrate that microalgal–bacterial interactions in this simplified model significantly influenced the architecture of their extracellular environment. We observed that the composition of the exo-environment, as described by the EPS monosaccharide profiles, varied under different culture conditions and times of incubation. This study reports an initial characterization of the molecular modifications occurring in the extracellular environment surrounding two relevant representatives of marine systems.     

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion. Received: 2 December 1996 / Accepted: 6 March 1997     

MOVEMENT MODALITIES AND RESPONSES TO ENVIRONMENTAL CHANGES OF THE MUDFLAT DIATOM CYLINDROTHECA CLOSTERIUM (BACILLARIOPHYCEAE)1

Cylindrotheca closterium (Ehrenberg) Reiman et Lewin is a raphid diatom widely distributed in mudflat assemblages. Video microscopy showed various movement modalities defined as smooth and corkscrew gliding, pirouette, pivot, rock and roll, rollover, and simultaneous pirouette and gliding. Z‐axis projection analysis of images revealed a unique gliding motif with corkscrew motions, which may have important ecological implications for C. closterium movement in muds. The general response to salinity alteration was a decrease in gliding movements with a concomitant increase in other modalities listed above. Short‐term responses to salinity change include dramatic alteration in modalities in hypo‐saline conditions and cessation of motility in extreme hyper‐saline environments. Modality changes were rapid and occurred within 5 s in response to hyper‐saline conditions. Hypo‐ or hyper‐saline conditions resulted in decreased gliding speed in standard media. Five‐ and 15‐day acclimation to salinity changes resulted in a progressive reduction in gliding movement, increased non‐gliding modalities and increased cell aggregation. Aggregation in hypo‐saline conditions was accompanied by a large increase in the polymer extracted by hot bicarbonate‐ and ethylenediamine tetraaceticacid‐ fractions of extracellular polymeric substance (EPS), the polymers of which have been implicated in cell attachment/motility phenomena. The monosaccharide profiles of these fractions were altered in response to hypo‐saline conditions. In general, monosaccharide profiles showed increased diversity upon cessation of motility and aggregation of cultures. The movement responses of C. closterium in response to environmental changes, accompanied by modifications in EPS, may form part of an adaptive strategy to survive in mudflats and could be useful as bioindicators of environmental changes.     

Photoluminescence shift in frustules of two pennate diatoms and nanostructural changes to their pores

The diatom silicified cell wall (frustule) contains pore arrays at the micro‐ to nanometer scale that display efficient luminescence within the visible spectrum. Morphometric analysis of the size and arrangement of pores was conducted to observe whether any correlation exists with the photoluminescence (PL) of two diatom species of different ages. UV‐excited PL displays four clearly defined peaks within the blue‐region spectrum, on top of the broad PL characteristic of synthetic porous silicon dioxide, recorded for reference and where discrete lines are absent. A set of shifted emission lines was observed when diatom cultures reached adulthood. These discrete line shifts correlate with structural changes observed in adult frustules: reduction in pore diameter; appearance of pores within pores, 10 nm in size; an increase in the gap distance between stria; and the deposition of several girdle bands with a concomitant increase in the diatom waist length, as well as the appearance of pores on such bands. Destruction of the pores results in the disappearance of all discrete emission lines. The PL shifts are correlated with a substantial increment of Si–OH groups adsorbed on the frustule surface, as revealed by Fourier transform infrared spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Utilizing QCM-D to characterize the adhesive mucilage secreted by two marine diatom species in-situ and in real-time

The quartz crystal microbalance with dissipation monitoring (QCM-D) was used to monitor the deposition of adhesive extracellular polymeric substances (EPS) employed by the marine biofouling diatoms Craspedostauros australis Cox and Amphora coffeaeformis Cleve during initial adhesion and subsequent motility. Upon injection into the QCM chamber, initial negative frequency (f) shifts and positive dissipation (D) shifts were measured that correlated to cells impacting and adhering to the QCM sensor surface. Following this "initial adhesion" response, f continued to decrease while D increased logarithmically. Rather than the result of any cell morphological alterations at the substrate surface, the shifts were correlated to the time-dependent deposition of EPS upon the substrate surface as a result of cellular motility, or gliding. Experiments utilizing comparable cell concentrations of the diatom species C. australis and A. coffeaeformis revealed significant differences between the parameter responses recorded, where A. coffeaeformis produced Deltaf and DeltaD values of -32 Hz and 6.6, and C. australis produced values of -82 Hz and 42, respectively, after 20 h post-inoculation. The viscoelastic properties of the adhered EPS adlayer from both species were examined via a Deltaf/DeltaD plot, providing reproducible signature "ratio" values for each species that likely correlate to differences in EPS interactions with the substrate that may be associated directly to differences in the fouling potential of the two species. There is a distinct lack of knowledge regarding the chemical nature of the adhesive polymers engaged, and few quantitative techniques are applicable to the study of diatom EPS. We propose that QCM-D may be a useful tool in identifying differences in the EPS employed by diatoms of different fouling potential.     

Fine structure and 18S rDNA phylogeny of a marine araphid pennate diatom Plagiostriata goreensis gen. et sp. nov. (Bacillariophyta)

A marine araphid pennate diatom Plagiostriata goreensis is described from the sand grains of Goree Island, Dakar, Republic of Senegal, based on observations of fine structure of its frustule. The most striking feature of the species is its striation, which is angled at approximately 60° across the robust sternum. The other defining features of the species are its one highly reduced rimoportula and apical pores located at both ends of the valve margin. In the 18S rDNA phylogeny, the species appears as a member of a ‘small‐celled clade’ of araphid pennate diatoms that consist of Nanofrustulum, Opephora and Staurosira. The results of the phylogenetic analyses suggest that the distinct characters of the diatom; namely, oblique striae and apical pores, may have been acquired independently. However, it remains unclear whether the rimoportula of P. goreensis is a reduced state or P. goreensis acquired its morphologically curious rimoportula independently after the loss of an ancient rimoportula at the root of the small‐celled clade.     

COMPOSITION OF EXTRACELLULAR POLYMERIC SUBSTANCES FROM PERIPHYTON ASSEMBLAGES IN THE FLORIDA EVERGLADES1

In wetland habitats, periphyton is a common component of open‐water areas with species assemblage determined by local water quality. Extracellular polymeric substances (EPS) secreted by algae and bacteria give structure to periphyton, and differences in EPS chemistry affect the functional roles of these polymers. The Florida Everglades provide a unique opportunity to study compositional differences of EPS from distinctive algal assemblages that characterize areas of differing water chemistry. Water conservation area (WCA)‐1 is a soft‐water impoundment; periphyton was loosely associated with Utricularia stems and amorphous in structure, with a diverse desmid and diatom assemblage, and varying cyanobacterial abundance. Extracellular polymers were abundant and were loosely cell‐associated sheaths and slime layers in addition to tightly cell‐associated capsules. The EPS were complex heteropolysaccharides with significant saccharide residues of glucose, xylose, arabinose, and fucose. Carboxylic acids were also prominent, while ester sulfates and proteins were small components. Structured, cohesive cyanobacteria‐dominated periphyton was observed in WCA‐2A, a minerotrophic impoundment, and filaments were heavily encrusted with calcium carbonate and detrital matter. EPS were primarily cell‐associated sheaths, and polymer residues were dominated by glucose, xylose, fucose, and galactose, with uronic acids also a significant component of the polymers. Principal components analysis revealed that periphyton community assemblage determined the monosaccharide composition of EPS, which ultimately determines a range of biogeochemical processes within the periphyton.     

Influence of bacteria on cell size development and morphology of cultivated diatoms

Vegetative cell division in diatoms often results in a decreased cell size of one of the daughter cells, which during long‐term cultivation may lead to a gradual decrease of the mean cell size of the culture. To restore the initial cell size, sexual reproduction is required, however, in many diatom cultures sexual reproduction does not occur. Such diatom cultures may lose their viability once the average size of the cells falls below a critical size. Cell size reduction therefore seriously restrains the long‐term stability of many diatom cultures. In order to study the bacterial influence on the size diminution process, we observed cell morphology and size distribution of the diatoms Achnanthidium minutissimum, Cymbella affiniformis and Nitzschia palea for more than two years in bacteria‐free conditions (axenic cultures) and in cultures that contain bacteria (xenic cultures). We found considerable morphological aberrations of frustule microstructures in A. minutissimum and C. affiniformis when cultivated under axenic conditions compared to the xenic cultures. These variations comprise significant cell length reduction, simplification and rounding of the frustule contour and deformation of the siliceous cell walls, features that are normally found in older cultures shortly before they die off. In contrast, the xenic cultures were well preserved and showed less cell length diminution. Our results show that bacteria may have a fundamental influence on the stability of long‐term cultures of diatoms.     

Diatom motility and low frequency electromagnetic fields--a new technique in the search for independent replication of results

The hypothesis that exposure to a certain combination of static and alternating electromagnetic fields (EMFs) results in an increase in motility of the marine diatom Amphora coffeaeformis was tested. Diatom motility in three strains of A. coffeaeformis was positively correlated with extracellular calcium ion (Ca2+) concentration. The test apparatus consisted of two pairs of Helmholtz coils supported around the stage of a microscope linked to a video recorder and monitor. This system allowed real-time in vivo recordings of diatom speed under EMF and control exposures. The EMFs were calculated at calcium resonance values, previously found to cause enhanced motility. Computerised image analysis was used to calculate the distance moved by individual diatoms in 2-min periods before, during and after EMF or sham-EMF (control) exposure. The addition of EMF caused no significant increase in diatom motility. The results are discussed in relation to the use of diatom motility to measure EMF exposure effects.